Cellular damage to aerobic organ tissues is well recognized as a consequence of ischemia, whether endogenous as in the case of spontaneous coronary artery occlusion, or iatrogenic such as with open heart, coronary bypass surgery, or transplant procedures with the hear or other organs such as the lung, liver, kidney, pancreas and gastrointestinal tract. The degree and duration of the ischemia causing events are relevant to the amount of cell death and/or reversible cellular dysfunction. It is also known that much of the tissue damage in fact occurs upon reperfusion (i.e. resumption of blood flow) and re-oxygenation of the previously anoxic tissue.
As a side product of normal aerobic respiration, electrons are routinely lost from the mitochondrial electron transport chain. Such electrons can react with molecular oxygen to generate the reactive free radical superoxide which through other reaction steps in the presence of hydrogen peroxide and iron produces the extraordinarily reactive and toxic hydroxyl radical. Metabolically active aerobic tissues possess defense mechanisms dedicated to degrading toxic free radicals before these reactive oxygen species can interact with cellular organelles, enzymes, or DNA, the consequences of which could, without such protective mechanisms, be cell death. These defense mechanisms include the enzymes superoxide dismutase (SOD) which disproportionates superoxide, catalase which degrades hydrogen peroxide, and the peptide glutathione which is a non-specific free radical scavenger.
While not fully understood, it is believed that with ischemia of metabolic tissues and subsequent reperfusion, a complex group of events occurs. Initially during the ischemic period, intracellular anti-oxidant enzyme activity appears to decrease, including that of SOD, catalase, and glutathione. There is also an indication that the level of xanthine oxidase activity concomitantly increases in vascular endothelial tissue during the ischemic event. The combination of enhanced ability to produce oxygen free radicals (via enhanced xanthine oxidase activity) and reduced ability to scavenge the same oxygen radicals (via reduced SOD, catalase and glutathione activity) greatly sensitizes the ischemic cell to an oxidative burst, and hence damage, should these cells be subsequently reperfused with blood and therefore oxygen. This oxidative burst occurring within seconds to minutes of reperfusion could result in reversible and irreversible damage to endothelial cells and other cells constituting the ischemic-reperfused organ matrix.
Peripheral vascular disease (PVD) is a common disease caused by atherosclerotic narrowing of the arteries of the lower extremities (Cooke, J. P., et al., Vasc. Med. 2:227–230 (1997); Brass, E. P. et al., Vasc. Med. 5:55–59 (2000); Hiatt, W. R., et al., IJCP Suppl. 119:20–27 (2001)). This disease affects 5% of all men and 2.5% of all women over the age of 60 in America (Weitz, J. I., et al., Circulation 94:3026–3049 (1996); Dawson, D. L. et al., Am. J. Med. 109:523–530 (2000)). In the United States, the prevalence of PVD is approximately 3 million and the incidence is 1 million new cases/year. Thus, the prevalence of this disease is increasing and will continue to increase as the population ages (Weitz, J. I. et al., supra).
Risk factors associated with PVD include age, hypertension, hyperlipidemia, hyperhomocysteinemia, hyperinsulinemia, insulin resistance, impaired glucose tolerance, smoking and diabetes mellitus. Of these, smoking and diabetes mellitus exhibit the strongest positive relationship to PVD.
Approximately a third of all patients diagnosed with PVD exhibit intermittent claudication, defined as lower extremity pain, muscle ache or muscle fatigue that is usually precipitated by exertion (Wood, A. J. J. et al., N. Engl. J. Med. 344:1608–1621 (2001)). Intermittent claudication results from ischemic disease of skeletal muscle characterized by repeated bouts of ischemia-reperfusion. In many ways, intermittent claudication resembles angina. In fact, patients presenting with intermittent claudication have a 3-fold higher incidence of cardiovascular mortality, and as many as 10% have co-existing cerebrovascular disease and 28% exhibit symptoms of coronary artery disease.
The all-cause, 5-year mortality in patients with intermittent claudication is 30% and if complicated by concomitant coronary artery disease, the mortality is 40%. The most serious complication of severe intermittent claudication is amputation of the affected limb, which is necessary in about 5% of all patients. Most often, amputation is required when non-healing ischemic ulcers on the affected limb become infected and/or gangrene occurs.
An underlying metabolic disorder of energy generation develops in ischemic skeletal muscle, which is not affected by revascularization or vasodilating agents. During normal aerobic metabolism, muscle tissue utilizes free fatty acids (FFA) to generate energy. During ischemia, the muscle switches to anaerobic metabolism, where glucose becomes the primary source of energy. Glucose oxidation consumes less energy than FFA oxidation, and hence glucose oxidation increases muscle efficiency during ischemia. However, the switch to glucose metabolism during ischemia is usually incomplete and some FFAs are utilized as a source of energy. This is due to the fact that glycolysis is inhibited in the presence of insulin resistance and high glucagon levels. In particular, excess blood FFA oxidation creates highly toxic free radicals that cause muscle tissue damage.
During ischemia, anaerobic glycolysis is an important source of ATP. Under these circumstances, glycogen is depleted and lactic acid accumulates. In low flow ischemia, such as occurs with narrowing of blood vessels to the lower extremities, the efficiency of ATP production is especially critical. In such an ischemic condition, the supply of oxygenated blood is limited but not absent, as may occur with thrombosis of a blood vessel resulting in complete obstruction. Fatty acid oxidation is inherently less efficient than glucose oxidation as a process for generating ATP. In fact, 8 to 50% more molecular oxygen is required to produce one molecule of ATP when fatty acids are used as the fuel source. Thus, in the setting of reduced blood flow, where oxygen delivery is limited, a more efficient means of producing ATP is glucose oxidation. Insulin promotes glucose oxidation and reduces fatty acid oxidation by activating pyruvate dehydrogenase (PDH), by enhancing glucose transport into muscle and by inhibiting fatty acid oxidation. The therapeutic benefit of administering insulin with glucose to patients suffering from muscle ischemia has been demonstrated in both animals and humans. However, this combination therapy creates an imbalance between blood glucose and insulin levels causing the patients to become hyperglycemic or hypoglycemic. These major adverse effects limit the utility of glucose plus insulin as a therapeutic combination.
As a side product of normal aerobic respiration, electrons are routinely lost from the mitochondrial electron transport chain. Such electrons react with molecular oxygen to generate the reactive free radical superoxide, which through a series of reaction steps, in the presence of hydrogen peroxide and iron, produces the extraordinarily reactive and toxic hydroxyl radical. Metabolically active aerobic tissues possess defense mechanisms including superoxide dismutase (SOD), which removes the free superoxide anion radical O2−; catalase, which degrades hydrogen peroxide; and glutathione, which scavenges free radicals; all of which are dedicated to degrading toxic free radicals. In the absence of such defense mechanisms these reactive oxygen species interact with cellular organelles, enzymes, or DNA, ultimately causing cell death.
A complex series of events occurs during ischemia and subsequent reperfusion of metabolic tissue. During the ischemic period, intracellular anti-oxidant enzyme activity, including that of SOD, catalase, and glutathione, decreases with a concomitant increase in xanthine oxidase activity in vascular endothelial tissue. The combination of an enhanced ability to produce oxygen free radicals (via enhanced xanthine oxidase activity) and reduced ability to scavenge the same oxygen radicals (via reduced SOD, catalase, and glutathione activity) greatly sensitizes the ischemic cell to an oxidative burst. Reperfusion of the tissue results in an oxidative burst within seconds to minutes of the reperfusion. The oxidative burst can result in damage to endothelial cells and other cells constituting the ischemic-reperfused tissue.
Attendant with the initial oxidative burst is oxidation damage to cell membranes. Lipid oxidation in cell membranes causes neutrophil chemotaxis to post-ischemic areas. Such activated neutrophils adhere to the vascular endothelium, induce the conversion of xanthine dehydrogenase to xanthine oxidase within the endothelial cells, and further aggravate the loss of endothelial integrity. Activated neutrophils also migrate out of the vasculature where they can directly kill myocytes.
Additional consequences of ischemia-reperfusion include reversible skeletal muscle dysfunction resulting from perturbations in normal calcium mobilization from the sarcoplasmic reticulum.
The paradox of cellular damage associated with a limited period of ischemic anoxia followed by reperfusion is that cell damage and death appear not only to directly result from the period of oxygen deprivation but also as a consequence of re-oxygenating the tissues. Reperfusion damage commences with the initial oxidative burst immediately upon reflow and worsens over a number of hours as inflammatory processes develop in the same post-ischemic tissues. Efforts dedicated to decreasing sensitivity of post-anoxic cells to oxidative damage and, additionally, efforts to reduce inflammatory responses in these same tissues have been shown to reduce the damage to post-anoxic reperfused tissue.
It, therefore, can be seen that there is a need for a safe effective composition having broad applicability to prevent or ameliorate the harmful effects of ischemia and reperfusion for tissues in general.
Three types of therapy for intermittent claudication are currently available, depending upon the severity and extent of the disease. For less severe cases, medical management consists of risk factor management, exercise training and chronic therapy with anti-platelet drugs (e.g., aspirin, ticlopidine, or clopidogrel) or vasodilators (e.g., pentoxifylline and cilostazol). Patients with more severe symptoms may undergo peripheral angioplasty or intravascular stent placement. Surgical revascularization is reserved for limb-threatening ischemia that is anatomically amenable to operation. Thus, there is a need for better methods for treating or preventing intermittent claudication.